Cryopreservation: An Overview of Principles and Cell-Specific Considerations

Abstract

The origins of low-temperature tissue storage research date back to the late 1800s. Over half a century later, osmotic stress was revealed to be a main contributor to cell death during cryopreservation. Consequently, the addition of cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), or propylene glycol (PG), although toxic to cells at high concentrations, was identified as a necessary step to protect against rampant cell death during cryopreservation. In addition to osmotic stress, cooling and thawing rates were also shown to have significant influence on cell survival during low temperature storage. In general, successful low-temperature cell preservation consists of the addition of a CPA (commonly 10% DMSO), alone or in combination with additional permeating or non-permeating agents, cooling rates of approximately 1ºC/min, and storage in either liquid or vapor phase nitrogen. In addition to general considerations, cell-specific recommendations for hepatocytes, pancreatic islets, sperm, oocytes, and stem cells should be observed to maximize yields. For example, rapid cooling is associated with better cryopreservation outcomes for oocytes, pancreatic islets, and embryonic stem cells while slow cooling is recommended for cryopreservation of hepatocytes, hematopoietic stem cells, and mesenchymal stem cells. Yields can be further maximized by implementing additional pre-cryo steps such as: pre-incubation with glucose and anti-oxidants, alginate encapsulation, and selecting cells within an optimal age range and functional ability. Finally, viability and functional assays are critical steps in determining the quality of the cells post-thaw and improving the efficiency of the current cryopreservation methods.

Aquaporins and Animal Gamete Cryopreservation: Advances and Future Challenges

“…CPAs are generally classified as penetrating or nonpenetrating, according to their plasma membrane permeability [23]. Penetrating CPAs are commonly organic compounds characterized by a low molecular weight (generally less than 100 Dalton) and high amphiphilic properties, which confers permeability through the plasma membrane [32]. The most well-known and used penetrating CPAs are glycerol, ethylene glycol, propylene glycol (1,2-propanediol), dimethyl sulfoxide (DMSO), methanol, and butanediol (characteristics and toxicity are extensively reviewed by Elliott et al [31]).…”

Cryopreservation Methods and Frontiers in the Art of Freezing Life in Animal Models

“…Henceforth, they protect the cell from intracellular ice formation (IIF) and salt accumulation. Examples of CPAs in this category are: glycerol (the first agent discovered), dimethyl sulfoxide (DMSO), ethylene glycol (EG), and propanediol (propylene glycol) [39]. The protective role of CPAs is due to hydrogen bonding with water molecules, and lowering the freezing point of water.…”

“…This can be explained thermodynamically as vitrified state is quasistable and can change into a lower energy crystal structure on thawing. Currently, to achieve maximum viability it is suggested to transport cryovials on vapor LN 2 and warmed rapidly in a 37°C water bath for 90-120 s [39]. Decrease in viability after post thaw recovery is inevitable, no matter how well the cells were stored and thawed.…”

Oocyte Cryopreservation in Domestic Animals and Humans: Principles, Techniques and Updated Outcomes

“…In addition, penetrating CPAs also help to increase the hydration status of cells during cooling in order to prevent the excessive accumulation of cellular electrolytes. Penetrating CPAs have, in general, a molecular weight typically less than 100 Daltons and with high amphiphilic properties [39]. However, excessive accumulation of penetrating CPAs also results in an increased cellular toxicity.…”